Stem cells are undifferentiated cells which exhibit the capacities for self-renewal and differentiation into more than two kinds of mature somatic cells. They are classified into totipotent stem cells, pluripotent stem cells, multipotent stem cells and bipotent stem cells according to their differentiation capacity. Furthermore, they are also classified into embryonic stem cells (ESCs), hematopoetic stem cells, mesenchymal stem cells and induced pluripotent stem cells (iPSCs) upon their origins. Herein, hESCs, derived from the inner cell mass of the human blastocyst, have shown the pluripotency for differentiating into all somatic cells. In addition, iPSCs, reprogrammed from differentiated somatic cells by introducing Yamananka factors or certain pluripotent factors, show pluripotency in vitro and in vivo and share ESC-like characteristics.
Many investigators have focused on the control of the stem cells differentiation into specific cell lineages with high efficiency. They attempt to apply these stem cells or their derivative cells to injured tissues for recovery of normal physiological functions. For example, the hESC-derived retina pigment epithelial (RPE) cells have been enriched in vitro and used to rescue the eye sight of patients with Stargardt Macular dystrophy and age-related macular degeneration after the cell transplantation.
Neural stem cells and stem cells-derived neurons benefit biomedical investigations in studying neural development, neural physiology, and development of new therapy for treating neural trauma and neuro-degeneration diseases. Therefore, robustly producing neuroepithelial cells, the most primitive precursor cells of embryonic nerve system, is highly potential for the above applications.
Many investigators have tried to induce neural differentiation of ESCs by adding fibroblast growth factor-2 (FGF-2) at the earliest step of differentiation under suspension culture (Li, X. J. et al., 2005; Timothy et al., 2009; Xu et al., 2005; Vallier, L. et al., 2005). Although this FGF2-dependent neural induction method can trigger the differentiation of ESCs into the neuroepithelial cells and show the cellular expression profile as the cells in neural groove of embryo, it usually spends 10-14 days for this stage of neural induction. In addition, some pluripotent stem cells are refractory to this method and show low efficacy of neural production. The low purity of ESC-derived neural cells renders non-neural cells contamination within the cultured population and hinders further clinical applications.
Dual Smad inhibition method has shown the high efficacy of neuroepithelial cell induction from pluripotent stem cells. Inhibitors of Smad 1/5/8 and Smad2/3, such as Noggin and SB431542 respectively, were added into the induction medium for shortening the time frame of the neural induction (Elkabetz et al., 2008; Lee et al., 2007; Chambers et al., 2009). In addition, genetic manipulations or co-culture with other cell lines were also reported to induce the neural differentiation of pluripotent stem cells.
Although the aforementioned methods for generating the neuroepithelial cells from pluripotent stem cells are available, their disadvantages, such as poor efficacy of neural differentiation, expansive cost, risks resulted from virus-mediated genetic manipulations and non-neural cells contamination, still limit the further application of neuroepithelial cells in clinics. In addition, the contaminated non-neural cells or undifferentiated stem cells in the cultured pool may affect further neural differentiation. Moreover, remaining undifferentiated pluripotent stem cells may bring the risk of teratoma formation if the cells are not cleared before transplantation into recipients.
Therefore, efficient production of primitive neuroepithelial cells with high purity will benefit the further differentiation into mature neural cells and improve the clinical reliability and safety.